Ateneo - UPO

Significance of the measurement in the biological field.

Sample preparation techniques.

Principles of spectrophotometry.

Principles underlying chromatography.

Principles underlying electrophoresis.

Techniques for protein characterization.

Applications of antibodies in biotechnology.

Use of enzymes in diagnostics and research.

Binding and association assays.

"Introduzione alla biochimica di Lehninger"

David L. Nelson & Michael M. Cox

Zanichelli

Wet laboratory experience aims to provide the practical skills required to use basic biochemical techniques and to succeed in a profitable thesis internship.

The laboratory theory lessons present advanced techniques that students encounter in the scientific literature.

Students MUST have successfully attended the "safety in chemical and biological laboratories" course.

The knowledge on the structure and properties of molecules provided by inorganic and organic chemistry is preparatory.

The course includes:

- lectures on the main techniques

- computer simulations of the main techniques used in biochemistry laboratories.

- exercises in chemical-biological didactic laboratories.

Attendance is mandatory. To obtain a laboratory grade, students must have attended at least 75% of the lectures (i.e., at least 22 of the 30 total hours of the course).

At the end of the practical laboratory, each student will submit a laboratory notebook, completed during the activities, documenting the procedures performed and their understanding of the content. A similar report will be submitted at the end of the "virtual" exercises conducted using simulation software.

On the final day of the practical laboratory, a skills assessment will be held to assess the student's understanding of the concepts covered in the lectures and exercises. The test will include multiple-choice questions, open-ended questions, and application exercises.

The final grade will be based on the integration of the test results with the quality of the report and laboratory notebook.

Students with physical disabilities, Specific Learning Disorders (SLD), or Special Educational Needs (SEN) can request specific services and tools dedicated to them by contacting the Student Development and Career Coordination Staff and the Student Services team, and by consulting the dedicated page on the university's website: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities

Students with disabilities, DSA, BES, once they have contacted the University Staff, can reach out to the course instructor regarding the exam procedures and didactic aspects.

All students who have attended the theoretical lessons and the laboratory will be evaluated based on the results of the online exercises, the quality of their laboratory notebooks, and their performance in the laboratory.

Understanding the laboratory, instrumentation, measurement, dilution, and preparation of a calibration curve.

Using Excel to construct a calibration curve and practical exercises.

How to write a lab notebook.

How to read and interpret a protocol.

Theory and practical exercises on preparing solutions.

Meaning of the measurement in the biological field: specificity, accuracy, precision, and reproducibility.

Techniques for preparing samples for biochemical analysis (e.g., homogenization, detergent-based lysis). Extraction with solvents and selective precipitation, their application to the fractionation of biological compounds (lipid extraction, salting out of proteins and nucleic acids). Theory of centrifugation and preparative centrifugation techniques for fractionating biological compounds. Use of centrifuges and centrifugal force.

Principles of spectrophotometry. Theoretical foundations and practical aspects of spectroscopic techniques (absorbance, fluorescence, polarimetry), with examples of their use in quantifying biological analytes. Characteristics of a spectrophotometer and a fluorimeter, and their applications.

Principles of chromatography; main chromatographic techniques (affinity, gel filtration, ion exchange, and reversed-phase); and related matrices. Use of chromatography for separating complex mixtures of proteins and lipids based on their chemical and physical properties. Equipment used (column chromatography, thin-layer chromatography, HPLC, etc.). Detectors and analysis of results (retention coefficients, qualitative and quantitative analysis).

Principles of electrophoresis, with particular attention to protein fractionation and characterization. IEF, SDS-PAGE, 2D-PAGE, and capillary electrophoresis. Common detection systems: dyes and western blotting.

Applications of antibodies in biotechnology, competitive and non-competitive immunoassays, RIA, ELISA, immunofluorescence, western blotting, immunodiffusion, and related techniques. Use of enzymes in diagnostics and research. Characterization of enzymatic kinetics and study of inhibitors.

Use of enzymes in clinical diagnostics, kinetic and end-point assays applied to research and diagnostics. Analysis of enzymatic kinetic parameters.

The student will acquire knowledge of the main biochemical methodologies with particular emphasis on the study of proteins. Such knowledge is useful for the critical reading of technical literature.

The exercises allow you to move fruitfully in the laboratory, also in view of the degree internship. For this purpose, the practice of compiling the laboratory notebook is important.